This invention includes recombinant genetic methods to identify specific inhibitors of the purine nucleotide synthesis enzyme, inocsine 5'-monophosphate- dehydrogonase (IMPDH); an enzyme with favorable therapeutic potential in a variety of clinical settings.
Developments in combinatorial chemistry allow the rapid and economical synthesis of hundreds to thousands of discrete compounds. These compounds are typically arrayed in moderate-sized libraries of small organic molecules designed for efficient screening. Combinatorial methods, can be used to generate unbiased libraries suitable for the identification of novel inhibitors. In addition, smaller, less diverse libraries can be generated that are descended from a single parent compound with a previously determined biological activity. In either case, the lack of efficient screening systems to specifically target therapeutically relevant biological molecules produced by combinational chemistry such as inhibitors of important enzymes hampers the optimal use of these resources.
Inhibitors of the enzyme IMPDH (EC 1. 1. 1.205) which catalyzes the formation of xanthine monophosphate (XMP) from inosine monophosphate (IMP), are sought -for clinical use. In the purine de novo synthetic pathway, IMPDH is positioned at the branch point in the synthesis of adenine and guanine nucleotides and is, thus, the rate-limiting enzyme of guanine nucleotide biosynthesis. The guanine nucleotide products of the purine de novo pathway are essential precursors for DNA and RNA biosynthesis, Guanine nucleotides are also an important component of vital cellular processes such as glycosylation, signal transduction, and regulation of metabolic pathways. Because of these essential functions involving the activity of IMPDH, all free living organisms contain the necessary genetic information to produce the IMPDH enzyme. At the present time, IMPDH coding regions have been identified for greater than 25 different species with representatives from the eukaryotic, prokaryotic, and archaeal domains.
Because IMPDH is essential in providing the necessary precursors for DNA and RNA biosynthesis, normal tissues that exhibit increased cell proliferation generally exhibit increased IMPDH activity. Similarly, increased cell proliferation is accompanied by elevated enzyme activity in certain rat hepatomas with varied growth rates. These hepatomas manifest IMPDH activities that are disproportionately higher than those of normal tissues, suggesting that IMPDH is associated with cell proliferation and may be linked to either malignant cell transformation or tumor progression. Conversely, inhibiting IMPDH activity should restrict cell proliferation and induce toxicity or cell differentiation.
The essential nature of IMPDH is also reflected in the diverse applications of IMPDH inhibitors in the areas of cancer chemotherapy, viral infections, immunosuppression, and autoimmune diseases. Several IMPDH inhibitors are being evaluated for their utility as antineoplastic agents. One of these, tiazofurin, has undergone Phase-I/II studies to assess its efficacy against end stage leukemia in adult patients (Jayaram et al., 1992) and is moderately effective in inducing clinical remissions. Mizoribine and mycophenolate mofetil are both inhibitors of mammalian IMPDH useful for immunosuppression following organ transplantation (Halloran et al., 1996-1 Hughes et al., 1996; Allison et al., 1996). The immunosuppressive effects of IMPDH inhibitors would be useful in the treatment of chronic inflammatory diseases such as arthritis, diabetes or systemic lupus erythromatosis.
Another IMPDH inhibitor, ribavirin, is an antiviral agent used to treat respiratory syncytial virus (RSV) infections in pediatric patients (Smith et al., 1991) and for the treatment of AIDS (Japour et al., 1996).
These reports demonstrate the therapeutic value IMPDH inhibitors in a variety of clinical settings. The rapid accumulation of sequence information from various organisms will provide new IMPDH enzymes as potential therapeutic targets. Although there are several effective inhibitors of mammalian IMPDH enzymes, the utility of IMPDH inhibitors as antimicrobial, antifungal or antiparasitic therapeutic agents has not been widely investigated. These applications are important in view of the rapid spread of antibiotic, antifungal, and antiprotozoal resistance (Gillespie, 1997; Klepser 1997). The emergence of opportunistic pathogens, especially in the immunocompromised host, and the widespread use of antibiotics have resulted in serious problems in treating infectious diseases.
Several effective inhibitors of human IMPDH such as mycophenolic acid (MPA) or ribavirin have been identified by nonsystematic methods. Such inhibitors of the human enzyme already have a demonstrated utility in several therapeutic modalities (Table 1). A method to identify new inhibitors of human IMPDH with improved selectivity or bioavailability would enhance the clinical utility of these therapeutic agents. In addition, investigation of the clinical or veterinary utility of IMPDH inhibitors as antimicrobial, antifungal or. antiprotozoal agents would be facilitated by a rapid screening assay. These applications are important in view of the rapid spread of resistance to antimicrobial drugs. The emergence of opportunistic pathogens, especially in immunocompromised hosts, and the widespread use of antibiotics have resulted in serious problems in treating infectious diseases. In view of the need for new antimicrobial agents and the promising potential of IMPDH as a therapeutic target, new methods to efficiently identify IMPDH inhibitors would greatly expedite/enhance the clinical or veterinary applications of such agents.
TABLE 1 ______________________________________ Clinically Useful Inhibitors of IMPDH Inhibitor Clinical Application Publications ______________________________________ Tiazofurin Cancer therapy Jayaram et al., 1992 Mizoribine (Bredinin) Immunosuppression Halloran et al., 1996 Mycophenolate mofetil Immunosuppression Allison et al., 1996 Ribavirin Antiviral therapy Smith et al., 1991 Ribavirin AIDS therapy Japour et al., 1996 ______________________________________
There are fundamental differences in sensitivity to inhibitors and in kinetic parameters among the various IMPDH enzymes from eukaryotic, prokaryotic, and archaeal sources. Information in Table 2 was compiled from unpublished results of the inventors and the data in Hagen et al., 1995. Therefore, a large number of candidate inhibitors should be screened to identify the most effective one for each application. However, there is not a procedure to systematically screen the various chemical agents for utility as IMPDH inhibitors. Therefore methods are needed for identifying new specific IMPDH inhibitors
TABLE 2 ______________________________________ Comparison of IMP Dehydrogonase Sequences from Eukaryotic, Prokaryotic and Archaeal Sources. Mycobactefium Pyrococcus Characteristic Human tuberculosis Furiosus ______________________________________ Molecular Weight(KDa) 56 54.7 52.9 Isoelectric Point(cal) 7.1 6.1 5.9 IMP-K.sub.m (:M) 20 50 -- AND-K.sub.m (:M) 35 900 50 MPA-K.sub.j (nM) 20 No inhibition Slight inhibition ______________________________________
IMPDH isolated from bacterial sources has been determined to vary widely with respect to allosteric properties, size, and subunit composition. IMPDH isolated from E. coli has been purified and characterized as a tetramer of identical subunits. Unlike mammalian enzymes, the E. coli IMPDH is reported to be insensitive to the inhibitory effect of the mammalian inhibitor, mycophenolic acid. In E. coli, IMPDH has been determined to be the product of the guaB locus. The sequence of the guaB structural gene and surrounding DNA has been determined to span 1.533 Kb and to code for an IMPDH subunit of 511 amino acids with a calculated molecular mass of 54,512.
The Bacillus subtilis IMPDH gene has been cloned and, upon reintroduction into a B. subtilis strain that overproduced inosine resulted in an increased production of guanosine, accompanied by a decreased accumulation of inosine. The IMPDH gene was localized on a 6.5-Kb insert and further localized to a Hind III-partially digested 2.9-Kb fragment. However, the gene was not reported to have been isolated and no information was provided with respect to the DNA sequence of the gene.
In summary, no general, efficient screening system for IMPDH inhibitors is available. Development of such an assay would enhance and accelerate the discovery of therapeutically useful inhibitors.